Macrophages are target cells for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection that serve as viral reservoirs in brain, lung, gut, and other tissues, and play important roles in disease pathogenesis, particularly HIV/SIV-associated neurological disease. Macrophages express low levels of the HIV/SIV receptor CD4, but mechanisms by which macrophage-tropic viruses use low CD4 to mediate spreading infections are poorly understood. One mechanism involves enhanced envelope glycoprotein (Env) interaction with CD4 or CCR5, but this phenotype is frequently associated with increased neutralization sensitivity to antibodies targeting CD4/CCR5 binding sites. Moreover, this mechanism does not explain how these neutralization-sensitive viruses evade immune responses while establishing spreading infections. In this dissertation, we sought to identify SIV Env determinants for macrophage tropism and characterize mechanisms by which they enhance virus replication in macrophages. To identify viral variants capable of inducing macrophage-associated pathogenesis, we cloned Env sequences from SIV-infected macaques at early and late stage infection, and identified an early variant in blood that shares >98% sequence identity with the consensus sequence of late variants in brain from macaques with neurological disease. SIV clones encoding this Env variant mediated high levels of fusion, replicated efficiently in rhesus PBMC and macrophages, and induced multinucleated giant cell formation upon infection of macrophage cultures. We identified an N-linked glycosylation site, N173 in the V2 region, as a determinant of macrophage tropism. Loss of N173 enhanced SIVmac239 macrophage tropism, while restoration of N173 in SIVmac251 reduced macrophage tropism, but enhanced neutralization resistance to CD4/CCR5 binding site antibodies. SIVmac239 N173Q, which lacks the N173 glycosylation site, mediated CD4-independent fusion and cell-to-cell transmission with CCR5-expressing cells, but could not infect CD4-negative cells in single-round infections. Thus, CD4-independent phenotypes were detected only in the context of cell-cell contact. The N173Q mutation had no effect on SIVmac239 gp120 binding to CD4 in BIACORE and co-immunoprecipitation assays. These findings suggest that loss of the N173 glycosylation site increases SIVmac239 replication in macrophages by enhancing CD4-independent cell-to-cell transmission through CCR5-mediated fusion. This mechanism may facilitate escape of macrophage-tropic viruses from neutralizing antibodies, while promoting spreading infections by these viruses in vivo.

Les macrophages sont une cible cellulaire importante du VIH-1 et sont impliqués dans la propagation virale et la constitution du réservoir. Les patients infectés par le VIH-2 présentent un contrôle naturel de l'infection qui est généralement absent chez les patients infectés par le VIH-1. Nous avons étudié ici la relation entre les macrophages et le VIH-2 afin d'évaluer leur contribution à la physiopathologie de l'infection. L'assemblage de particules virales dans des macrophages dérivés de monocytes (MDM) infectés par le VIH-2 se fait au niveau de la membrane de compartiments internes semblables aux VCC documentés dans les MDM infectés par le VIH-1. Les VCC des MDM infectés par le VIH-1 et le VIH-2 partagent la même composition protéique, et la même morphologie. Contrairement à Gag du VIH-1, la protéine Gag du VIH-2 est absente du cytosol et presque exclusivement localisée dans les VCC, ce qui suggère que Gag du VIH-2 est rapidement transportée vers le VCC une fois synthétisée dans le cytosol. Les particules de VIH-2 produites de novo par les MDM peuvent mûrir, mais sont faiblement infectieuses et se transmettent inefficacement aux cellules T activés. Cette faible infectiosité n'est pas associée avec l'expression du facteur de restriction BST-2 et n'est pas non plus améliorée par une baisse des niveaux d'expression de BST-2 induite par Vpu. Nos données suggèrent que les macrophages infectés par le VIH-2 ne contribuent probablement pas à la production et à la dissémination du virus in vivo. Cependant, les macrophages infectés par le VIH-2 peuvent représenter une source potentielle d'antigènes viraux qui pourraient stimuler les réponses des cellules T spécifiques du virus. / Macrophages are an important cellular target of HIV-1 and are potentially involved in viral spreading and constitution of the viral reservoir. HIV-2-infected patients exhibit a natural virological control of the infection that is generally absent from HIV-1-infected patients. Here, we studied the relationship between macrophages and HIV-2 to approach their potential contribution to the physiopathology of HIV-2 infection. Viral particles assembly in HIV-2-infected monocyte-derived macrophages (MDMs) occurred at the limiting membrane of internal compartments similar to virus-containing compartments (VCCs) documented in HIV-1-infected MDMs. Indeed, VCCs from HIV-1 and HIV-2-infected MDMs shared protein composition, as seen by confocal microcopy, and morphology, as seen by electron microscopy. Strikingly, HIV-2 Gag was mostly absent from the cytosol and almost exclusively localized to the VCCs, whereas HIV-1 Gag was distributed in both locations, suggesting that HIV-2 Gag is rapidly transported to the VCC membranes once synthesized in the cytosol. HIV-2 particles produced de novo by MDMs can mature, but are poorly infectious and inefficiently transmitted to activated T cells. This low infectivity neither correlate with expression of the restriction factor BST-2, nor was improved by Vpu-induced down-modulation of BST-2 levels. Our data suggest that, HIV-2-infected macrophages are unlikely to contribute to viral production and dissemination in vivo. However, HIV-2-infected macrophages accumulate large amounts of intracellular virus that may represent a potential source of viral antigens that could stimulate virus specific T cell responses.